It is estimated that 42 million American adults and about 3 million middle and high school students smoke cigarettes. Clinical and pre-clinical studies show that fetal nicotine exposure occurring as a result of mothers' tobacco use during pregnancy is associated with premature birth, sudden infant death, and an increase in the risk for cognitive disabilities. Whereas maternal nicotine use and the consequent fetal nicotine exposure are undeniable concerns, in reality, smoking is more prevalent among men (21.6%) than women (16.5%). However, whether a father's use of tobacco products has consequences for his offspring is not clear. To address this unexplored issue, we developed a mouse model of paternal nicotine exposure, in which adult male mice were exposed to nicotine in drinking water. While the nicotine exposure was ongoing, the male mice were bred with drug nave female mice. Quite unexpectedly, we found that the offspring of the nicotine- exposed fathers had significant hyperactivity and attention deficits. Equally unexpectedly, although the hyperactivity was present in both the male and female offspring, the attention deficits were present only in the male offspring. Moreover, the expression of genes for dopamine receptors (dopamine is the neurotransmitter that regulates motor and cognitive function) in the frontal cortex and striatum (two brain regions together involved in motor and cognitive function) showed brain region-specific and sex-specific changes in the offspring descending from the nicotine-exposed male mice. These surprising and novel observations led us to investigate epigenetic modifications in the father's spermatozoal DNA as a potential mechanism underlying the transmission of the behavioral and molecular phenotypes from the nicotine-exposed father to his offspring. We found significant changes in global DNA methylation status and the methylation status of the dopamine D2 receptor in the fathers' spermatozoa. Finally, F2 generation (grandchildren of the nicotine-exposed F0 males) derived by crossing F1 males from nicotine-exposed fathers with drug nave females also showed sex-specific attention deficits and dopamine receptor gene expression changes in the brain, suggesting that the effects of nicotine exposure were evident in multiple generations of descendants. Based on these observations, we propose the hypothesis that offspring sired by nicotine-exposed fathers show significant and sex-specific behavioral and molecular phenotypes, and that nicotine-induced epigenetic modification of the DNA of the fathers' spermatozoa is a mechanism for heritability of the phenotypes. We will test this hypothesis by performing behavioral analyses, whole-genome bisulfite sequencing to assay methylation status of the DNA, and RNA sequencing to assay changes in the transcriptome. The proposed studies offer excellent training opportunities for undergraduate and graduate students and strengthen the research base at our institution. !